A mechanism of carbapenem resistance due to a new insertion element (ISPa133) in Pseudomonas aeruginosa

This study explored the evolutionary mechanism by which the clinical isolate PA110514 yields the imipenemresistant derivative PA116136. Both isolates were examined by PFGE and SDS-PAGE, which led to the identification of a new insertion sequence, ISPa133. This element was shown to have distinct chromosomal locations in each of the original isolates that appeared to explain the differences in imipenem susceptibilty. In strain PA110514, ISPa133 is located 56 nucleotides upstream of the translational start codon, which has no effect on expression of the porin OprD. However, in strain PA116136 ISPa133 it is located in front of nucleotide 696 and, by interrupting the coding region, causes a loss of OprD expression, thus conferring imipenem resistance. In vitro experiments mimicking the natural conditions of selective pressure yielded imipenem-resistant strains in which ISPa133 similarly interrupted oprD. A mechanism is proposed whereby ISPa133 acts as a mobile switch, with its position in oprD depending on the degree of selective pressure exerted by imipenem

Avances en el estudio del "torrao" o cribado del tomate

El "torrao" es una enfermedad presente en nuestro país desde 2001, que sigue presentándose en cada campaña de tomate con mayor o menor incidencia según el año. Las plantas afectadas muestran necrosis en la parte basal de los foliolos que puede evolucionar a cribado, manchas longitudinales en los peciolos y manchas necróticas en fruto, que terminan por rajarlo. El presente trabajo es la continuación del publicado en el número 32 de esta revista titulado "Necrosis del tomate: "torrao" o cribado" y surge de los resultados obtenidos tras la reciente publicación de la identificación y caracterización del nuevo virus "Tomato torrado virus" (ToTV) como agente implicado en la enfermedad conocida como "torrao". En este estudio se seleccionaron 94 muestras procedentes de prospecciones realizadas en invernaderos de Murcia durante los años 2003 a 2006. La aplicación RT-PCR e hibridación molecular para la detección de ToTV ha permitido detectar la presencia de esta nueva virosis en 87 de las muestras analizadas. En 83 de ellas, se encontró la presencia conjunta de este nuevo virus con el Pepino mosaic virus (PepMV), mayoritariamente con el aislado tipo Chileno 2 (Accesión number: DQ00095). Se plantean nuevos estudios para determinar la implicación de ambos virus, ToTV y PepMV, en el desarrollo del síndrome conocido como "torrao" del tomate.

Necrosis del tomate: "torrao "o "cribado"

Desde la primavera de 2001, viene presentándose en España una nueva enfermedad conocida con el nombre de "torrao" o "cribado". Los síntomas que habitualmente presentan las plantas afectadas son una necrosis en la parte basal del foliolo que evoluciona a cribado, en los peciolos aparecen manchas longitudinales en ocasiones endurecidas que llegan a curvar los foliolos, y los frutos manifiestan manchas necróticas, deformaciones que finalmente lo rajan, quedando comercialmente inviables. Muestreos realizados desde su aparición han determinado la mayor incidencia de la enfermedad en la zona de Murcia, seguido de Canarias y en menor proporción Almería, y Alicante. Los resultados de los análisis realizados a las 369 muestras recogidas determinan que el 67% de las muestras analizadas eran positivas a Pepino mosaic virus (PepMV). En los ensayos de transmisión, únicamente mediante el injerto, se consiguió reproducir los síntomas de la enfermedad en dos casos, en el resto las plantas inoculadas e injertadas únicamente mostraban síntomas típicos de PepMV y los análisis realizados confirmaron este aspecto. A la vista de los resultados obtenidos, se diseñó un nuevo método de diagnóstico que ha permitido la caracterización del 89% de las muestras analizadas como aislado Chileno 2 de PepMV, recientemente publicado en el Gen Bank (Accesión number: DQ000985). De acuerdo con lo expuesto podría tratarse de uno de los agentes implicados en el desarrollo del síndrome junto con otros factores aún por determinar

Bioactividad de extractos de Melia azedarach L. sobre el taladro del maíz Sesamia nonagrioides Lef.

A partir de un extracto metanólico de semillas de Melia azedarach se han aislado los compuestos responsables de su actividad biológica como agentes inhibidores de la alimentación y reguladores del desarrollo sobre Sesamia nonagrioides. Las pruebas biológicas de actividad se han realizado incorporando cada fracción a la dieta, hojas de maíz o dieta sintética, de larvas de 2°estadio del insecto. La técnica de cromatografía líquida de alta resolución a escala semipreparativa, utilizando un cartucho de fase reversa, ha permitido separar dos componentes con cantidad y pureza suficientes para poder ser analizados con posterioridad mediante técnicas espectroscópicas.

Targeting Enterococcus faecalis Biofilms with Phage Therapy.

Phage therapy has been proven to be more effective, in some cases, than conventional antibiotics, especially regarding multidrug-resistant biofilm infections. The objective here was to isolate an anti-Enterococcus faecalis bacteriophage and to evaluate its efficacy against planktonic and biofilm cultures. E. faecalis is an important pathogen found in many infections, including endocarditis and persistent infections associated with root canal treatment failure. The difficulty in E. faecalis treatment has been attributed to the lack of anti-infective strategies to eradicate its biofilm and to the frequent emergence of multidrug-resistant strains. To this end, an anti-E. faecalis and E. faecium phage, termed EFDG1, was isolated from sewage effluents. The phage was visualized by electron microscopy. EFDG1 coding sequences and phylogeny were determined by whole genome sequencing (GenBank accession number KP339049), revealing it belongs to the Spounavirinae subfamily of the Myoviridae phages, which includes promising candidates for therapy against Gram-positive pathogens. This analysis also showed that the EFDG1 genome does not contain apparent harmful genes. EFDG1 antibacterial efficacy was evaluated in vitro against planktonic and biofilm cultures, showing effective lytic activity against various E. faecalis and E. faecium isolates, regardless of their antibiotic resistance profile. In addition, EFDG1 efficiently prevented ex vivo E. faecalis root canal infection. These findings suggest that phage therapy using EFDG1 might be efficacious to prevent E. faecalis infection after root canal treatment.

Occurrence of Arthroderma benhamiae Genotype in Japan.

In this study, we epidemiologically investigated on clinical isolates of Arthroderma benhamiae from humans and animals in Japan by internal transcribed spacer (ITS) region sequence analysis and mating type (MAT)-specific PCR. Seven of 8 A. benhamiae isolates from a human, rabbits and guinea pigs were identified as group I (white phenotype) by morphological characters and ITS region sequence analysis. One strain isolated from a degus (Octodon degus) produced colonies with few irregular folds and yellow velvety mycelium without macro- and microconidia. This strain resembled to group II (yellow phenotype) strain. ITS sequence analysis was also 100 % identical to that of group II. MAT-specific PCR indicated that 6 of these 7 isolates of group I contained an alpha-box gene and that one strain contained high-mobility-group (HMG) gene. One strain of group II was revealed to have an alpha-box gene and no HMG gene. To our knowledge, it is the first A. benhamiae isolate of group II found in Japan. The A. benhamiae may be more widespread in worldwide than our surpassing what is common or usual or expected.

Platinum (II) and palladium (II) complexes with (N,N') and (C,N,N') ligands derived from pyrazole as anticancer and antimalarial agents: synthesis, characterization and in vitro activities

The study of the reactivity of three 1-(2-dimethylaminoethyl)-1H-pyrazole derivatives of general formula [1-(CH2)2NMe2}-3,5-R2-pzol] {where pzol represents pyrazole and Rdouble bond; length as m-dashH (1a), Me (1b) or Ph (1c)} with [MCl2(DMSO)2] (Mdouble bond; length as m-dashPt or Pd) under different experimental conditions allowed us to isolate and characterize cis-[M{κ2-N,N′-{[1-(CH2)2NMe2}-3,5-R2-pzol])}Cl2] {MMdouble bond; length as m-dashPtPt (2a-2c) or Pd (3a-3c)} and two cyclometallated complexes [M{κ3-C,N,N′-{[1-(CH2)2NMe2}-3-(C5H4)-5-Ph-pzol])}Cl] {Mdouble bond; length as m-dashPt(II) (4c) or Pd(II) (5c)}. Compounds 4c and 5c arise from the orthometallation of the 3-phenyl ring of ligand 1c. Complex 2a has been further characterized by X-ray crystallography. Ligands and complexes were evaluated for their in vitro antimalarial against Plasmodium falciparum and cytotoxic activities against lung (A549) and breast (MDA MB231 and MCF7) cancer cellular lines. Complexes 2a-2c and 5c exhibited only moderate antimalarial activities against two P. falciparum strains (3D7 and W2). Interestingly, cytotoxicity assays revealed that the platinacycle 4c exhibits a higher toxicity than cisplatin in the three human cell lines and that the complex 2a presents a remarkable cytotoxicity and selectivity in lung (IC50 = 3 μM) versus breast cancer cell lines (IC50 > 20 μM). Thus, complexes 2c and 4c appear to be promising leads, creating a novel family of anticancer agents. Electrophoretic DNA migration studies in presence of the synthesized compounds have been performed, in order to get further insights into their mechanism of action.

Generalist hydrocarbon-degrading bacterial communities in the oil-polluted water column of the North Sea.

The aim of this work was to determine the effect of light crude oil on bacterial communities during an experimental oil spill in the North Sea and in mesocosms (simulating a heavy, enclosed oil spill), and to isolate and characterize hydrocarbon-degrading bacteria from the water column. No oil-induced changes in bacterial community (3 m below the sea surface) were observed 32 h after the experimental spill at sea. In contrast, there was a decrease in the dominant SAR11 phylotype and an increase in Pseudoalteromonas spp. in the oiled mesocosms (investigated by 16S rRNA gene analysis using denaturing gradient gel electrophoresis), as a consequence of the longer incubation, closer proximity of the samples to oil, and the lack of replenishment with seawater. A total of 216 strains were isolated from hydrocarbon enrichment cultures, predominantly belonging to the genus Pseudoaltero monas; most strains grew on PAHs, branched and straight-chain alkanes, as well as many other carbon sources. No obligate hydrocarbonoclastic bacteria were isolated or detected, highlighting the potential importance of cosmopolitan marine generalists like Pseudoalteromonas spp. in degrading hydrocarbons in the water column beneath an oil slick, and revealing the susceptibility to oil pollution of SAR11, the most abundant bacterial clade in the surface ocean.

Pricing of Product Variants

Työn tutkimusongelma oli selventää, tutkia ja analysoida dynaamisen hinnoittelun tekijät ja mahdollisuudet tuotevarianttien hinnoittelussa. Tutkimusongelman selvittämiseksi työlle asetettiin 8 tavoitetta - Saada selville miksi tuotevarianttien hinnoittelu on ongelmallista - Esittää kuinka tuotevarianttien hinnat teoreettisesti tulisi asettaa - Tunnistaa tuotevarianttien hinnoittelun ulottuvuudet ja selvittää dynaamisen hinnoittelun edut staattiseen hinnoitteluun verrattuna - Esitellä analyysikehikko hinnoittelun tilan analysointiin - Tunnistaa dynaamisen hinnoittelun tuotevarianteille suomat mahdollisuudet - Etsiä soveltuvat hinnoittelumenetelmät tuotevarianttien dynaamiseen hinnoitteluun - Analysoida tuotevariantteja myyvän yrityksen hinnoittelu - Tunnistaa ja arvioida dynaamisen hinnoittelun edut yritykselle Diplomityössä käytettiin useita tutkimusmenetelmiä. Perustieto haettiin kirjallisuustutkimuksella ja sitä täydennettiin haastatteluilla. Tutkimusprosessi alkoi tutkimuksella tuotevarianttien hinnoittelusta ja kirjallisuuden perusteella luotiin näkökulma ja yleiset kehityssuunnat tarkempaa tutkimusta varten. Kaksi tärkeintä tuotevariaatioiden hinnoitteludimensiota tunnistettiin ja niiden analysointia varten luotiin nelikenttämalli. Kirjallisuustutkimuksen ja tarkemman kohdeyrityksen tarkastelun perusteella dynaaminen tuotelinjahinnoittelu on tuotevarianttien dynaamisen hinnoittelun tavoitetila. Nelikenttämallia käytettiin kohdeyrityksen hinnoittelun tilan arviointiin ja dynaamisen hinnoittelun suurimmat hyödyt löydettiin. Tutkimuksen päätulokset ovat - Hinnoittelun dynaamisuutta tulee tuotevarianteilla tutkia hinnoittelun älykkyyden ja kehittyneisyyden kanssa - Tuotevariaatioiden hinnoittelun tavoitetila on dynaaminen tuotelinjahinnoittelu - Hinnoittelun kehittäminen staattisesta dynaamiseen tuo huomattavia etuja - Tärkein etu on parempi hintojen hallinta ja mahdollisuus johtaa hintoja tehokkaasti. Tämän vuoksi hinnoitteluanalyysissa havaittiin selvästi lisääntyneitä voittoja - Hinnoittelun älykkyyden nostaminen hyödyttää yritystä ja saa aikaan lisäyksen voitoissa

Characterization of gait in female patients with moderate to severe hallux valgus deformity.

BACKGROUND: Hallux valgus is one of the most common forefoot problems in females. Studies have looked at gait alterations due to hallux valgus deformity, assessing temporal, kinematic or plantar pressure parameters individually. The present study, however, aims to assess all listed parameters at once and to isolate the most clinically relevant gait parameters for moderate to severe hallux valgus deformity with the intent of improving post-operative patient prognosis and rehabilitation. METHODS: The study included 26 feet with moderate to severe hallux valgus deformity and 30 feet with no sign of hallux valgus in female participants. Initially, weight bearing radiographs and foot and ankle clinical scores were assessed. Gait assessment was then performed utilizing pressure insoles (PEDAR®) and inertial sensors (Physilog®) and the two groups were compared using a non-parametric statistical hypothesis test (Wilcoxon rank sum, P<0.05). Furthermore, forward stepwise regression was used to reduce the number of gait parameters to the most clinically relevant and correlation of these parameters was assessed with the clinical score. FINDINGS: Overall, the results showed clear deterioration in several gait parameters in the hallux valgus group compared to controls and 9 gait parameters (effect size between 1.03 and 1.76) were successfully isolated to best describe the altered gait in hallux valgus deformity (r(2)=0.71) as well as showed good correlation with clinical scores. INTERPRETATION: Our results, and nine listed parameters, could serve as benchmark for characterization of hallux valgus and objective evaluation of treatment efficacy.

Identification of Rare High-Avidity, Tumor-Reactive CD8+ T Cells by Monomeric TCR-Ligand Off-Rates Measurements on Living Cells.

The avidity of the T-cell receptor (TCR) for antigenic peptides presented by the peptide-MHC (pMHC) on cells is a key parameter for cell-mediated immunity. Yet a fundamental feature of most tumor antigen-specific CD8(+) T cells is that this avidity is low. In this study, we addressed the need to identify and select tumor-specific CD8(+) T cells of highest avidity, which are of the greatest interest for adoptive cell therapy in patients with cancer. To identify these rare cells, we developed a peptide-MHC multimer technology, which uses reversible Ni(2+)-nitrilotriacetic acid histidine tags (NTAmers). NTAmers are highly stable but upon imidazole addition, they decay rapidly to pMHC monomers, allowing flow-cytometric-based measurements of monomeric TCR-pMHC dissociation rates of living CD8(+) T cells on a wide avidity spectrum. We documented strong correlations between NTAmer kinetic results and those obtained by surface plasmon resonance. Using NTAmers that were deficient for CD8 binding to pMHC, we found that CD8 itself stabilized the TCR-pMHC complex, prolonging the dissociation half-life several fold. Notably, our NTAmer technology accurately predicted the function of large panels of tumor-specific T cells that were isolated prospectively from patients with cancer. Overall, our results demonstrated that NTAmers are effective tools to isolate rare high-avidity cytotoxic T cells from patients for use in adoptive therapies for cancer treatment.

Pineapple (Ananas comosus) cv. pérola ex vitro growth and mycorrhizal colonization affected by in vitro sucrose concentration

The aim of this study was to evaluate the effect of sucrose concentration in the culture medium on growth and on the establishment of mycorrhizas during the acclimatization of pineapple cv. Pérola. The plantlets were micropropagated in MS culture medium with 0, 10, 20 and 30 g L-1 of sucrose and then they were acclimatized during 12 weeks under greenhouse conditions, in a sandy soil - compost mixture, uninoculated or inoculated with a Rhizophagus clarus isolate. Plantlets from the culture medium with 20 g and 30 g of sucrose L-1 showed higher shoot and root biomass than those from sugar-free medium. Mycorrhizal colonization was lower in plantlets micropropagated in sucrose-free medium, but the intensity of arbuscules did not differ among treatments. In the 12-week period of acclimatization, mycorrhizal colonization had no effect on plant biomass.

Rockfall detection from LIDAR point clouds: A clustering approach using R

The analysis of rockfall characteristics and spatial distribution is fundamental to understand and model the main factors that predispose to failure. In our study we analysed LiDAR point clouds aiming to: (1) detect and characterise single rockfalls; (2) investigate their spatial distribution. To this end, different cluster algorithms were applied: 1a) Nearest Neighbour Clutter Removal (NNCR) in combination with the Expectation?Maximization (EM) in order to separate feature points from clutter; 1b) a density based algorithm (DBSCAN) was applied to isolate the single clusters (i.e. the rockfall events); 2) finally we computed the Ripley's K-function to investigate the global spatial pattern of the extracted rockfalls. The method allowed proper identification and characterization of more than 600 rockfalls occurred on a cliff located in Puigcercos (Catalonia, Spain) during a time span of six months. The spatial distribution of these events proved that rockfall were clustered distributed at a welldefined distance-range. Computations were carried out using R free software for statistical computing and graphics. The understanding of the spatial distribution of precursory rockfalls may shed light on the forecasting of future failures.

Knowing What to Respond in the Future Does Not Cancel the Influence of Past Events

Everyday tasks seldom involve isolate actions but sequences of them. We can see whether previous actions influence the current one by exploring the response time to controlled sequences of stimuli. Specifically, depending on the response-stimulus temporal interval (RSI), different mechanisms have been proposed to explain sequential effects in two-choice serial response tasks. Whereas an automatic facilitation mechanism is thought to produce a benefit for response repetitions at short RSIs, subjective expectancies are considered to replace the automatic facilitation at longer RSIs, producing a cost-benefit pattern: repetitions are faster after other repetitions but they are slower after alternations. However, there is not direct evidence showing the impact of subjective expectancies on sequential effects. By using a fixed sequence, the results of the reported experiment showed that the repetition effect was enhanced in participants who acquired complete knowledge of the order. Nevertheless, a similar cost-benefit pattern was observed in all participants and in all learning blocks. Therefore, results of the experiment suggest that sequential effects, including the cost-benefit pattern, are the consequence of automatic mechanisms which operate independently of (and simultaneously with) explicit knowledge of the sequence or other subjective expectancies.

A genotypic mutation system measuring mutations in restriction recognition sequences.

The RFLP/PCR approach (restriction fragment length polymorphism/polymerase chain reaction) to genotypic mutation analysis described here measures mutations in restriction recognition sequences. Wild-type DNA is restricted before the resistant, mutated sequences are amplified by PCR and cloned. We tested the capacity of this experimental design to isolate a few copies of a mutated sequence of the human c-Ha-ras1 gene from a large excess of wild-type DNA. For this purpose we constructed a 272 bp fragment with 2 mutations in the PvuII recognition sequence 1727-1732 and studied the rescue by RFLP/PCR of a few copies of this 'PvuII mutant standard'. Following amplification with Taq-polymerase and cloning into lambda gt10, plaques containing wild-type sequence, PvuII mutant standard or Taq-polymerase induced bp changes were quantitated by hybridization with specific oligonucleotide probes. Our results indicate that 10 PvuII mutant standard copies can be rescued from 10(8) to 10(9) wild-type sequences. Taq polymerase errors originating from unrestricted, residual wild-type DNA were sequence dependent and consisted mostly of transversions originating at G.C bp. In contrast to a doubly mutated 'standard' the capacity to rescue single bp mutations by RFLP/PCR is limited by Taq-polymerase errors. Therefore, we assessed the capacity of our protocol to isolate a G to T transversion mutation at base pair 1698 of the MspI-site 1695-1698 of the c-Ha-ras1 gene from excess wild-type ras1 DNA. We found that 100 copies of the mutated ras1 fragment could be readily rescued from 10(8) copies of wild-type DNA.